The prupose of this study is to determine how gene amplification occurs, and how other processes may alter the genome composition during nuclear differentiation in the simple eukaryote, Tetrahymena. We already know that rRNA gene exists as a single copy sequence in the micronuclear chromosome and is amplified to generate extrachromosomal copies in the macronucleus (14,18,22). Here I hope to determine how the first copy of extrachromosomal rDNA is produced during this process. The chromosomal rDNA and its neighboring sequences will be isolated from micronuclei by cloning in E. coli plasmids or in lambda phage. The sequence will be used to examine the organization of the corresponding sequences in the macronucleus by using restriction endonuclease gel electrophoresis and molecular hybridization techniques. The results should allow us to determine whether the integrated rDNA was excised out of the chromosome for amplification, and whether rejoining of the chromosome follows excision. Similar methods will be used to study other alteration processes. A small portion of the micronuclear DNA sequence has been found missing in the macronucleus (12). Recently we have detected in the genome many clusters of a repeated hexanucleotides (CCCCAA) which is closely associated with certain alteration processes. In this study I plan to isolate the sequences adjacent to the CCCCAA repeats from micronucleus by the cloning techniques, and determine whether chromosome breakage occurs near this repeated sequence, and whether the process leads to sequence elimination. The eliminated sequences will also be isolated from the micronucleus by the cloning techniques for further studies. This work should allow us to better understand the various events which occur normally to alter the constancy of a eukaryotic genome. It might also help us to interpret similar events which occur abnormally, like viral transformation.